WO2005012355A1 - Bioactive peptides derived from the proteins of egg white by means of enzymatic hydrolysis - Google Patents

Abstract

The invention relates to the production of ovoproducts containing bioactive peptides from the egg white subjected to enzymatic treatment. Said peptides have an inhibiting activity of the angiotensin converting enzyme (ACE inhibiting activity) in vitro and/or anti-hypertensive activity in rats and/or antioxidant activity. Said ovoproducts, complete hydrolizates, the fractions thereof with low molecular weight or their constituent peptides could be used as therapeutic substances with ACE inhibiting activity and/or anti-hypertensive activity and/or anti-oxidant activity, either as functional food products, food additives or ingredients or pharmaceutical products for the treatment and/or prevention of hypertension in all its forms in humans or animals and for the treatment and/or prevention of any disorder associated with hypertension in humans or animals.

Description

TITLE

Bioactive peptides derived from egg white proteins by enzymatic hydrolysis

TECHNICAL SECTOR The invention consists in the production of bioactive egg products derived from egg white proteins. These give rise, after an enzymatic treatment, to peptides with inhibitory activity of the angiotensin converting enzyme (TECA activity) in vitro and / or antihypertensive and / or antioxidant activity, which can be applied in the food and pharmaceutical industry.

STATE OF THE TECHNIQUE The role of the egg in human nutrition is essential as it constitutes a nutritious and healthy food. The recent development of new biotechnological and separation techniques allows the fractionation of different components of the egg to be used for new food or non-food purposes and, in this way, new applications are emerging that contribute to increasing its consumption. Thus, different companies dedicated to the production of isolated proteins from egg fractions are interested in increasing and diversifying the uses of some components, such as ovalbumin and ovotransferrin. This is the case of the industries dedicated to the production of lysozyme, used as an antimicrobial agent, which obtain by-products other low-cost nitrogenous fractions, generally intended for animal feed or for use as emulsifiers and gelling agents in different foods. In recent years, functional foods have broken into the food sector, due to the increased awareness of consumers of the relationship between diet and health. Within the functional ingredients, that is, components that, incorporated into the food, provide the same specific biological activities that go beyond mere nutrition, bioactive peptides occupy a prominent place, due to their diversity and multifunctionality. Bioactive peptides correspond to fragments that are inactive within the precursor protein, but which can be released by hydrolysis in vivo or in vitro and, in this way, exert different physiological functions in the body. Since its discovery, in 1979, peptides have been described food protein derivatives with different biological activities: antihypertensive, antithrombotic, opioid, antioxidant, immunomodulant, etc. Among the bioactive peptides, those that exert antihypertensive activity through the regulation of the renin-angiotensin system (T. Takano, Milk derived peptides and hypertension reduction, International Dairy Journal, 1998, 8: 375-381). The high incidence of coronary diseases in the population is notorious, and the treatment of hypertension is one of the most used strategies to reduce the risk of cardiovascular diseases. The mechanism of action of these peptides has been explained by the inhibition of the angiotensin-converting enzyme (RCT), which catalyzes the formation of angiotensin IL an octapeptide with a potent vasoconstrictor activity and, in addition, inactivates bradykinin, which produces vasodilation. Different peptides with ACE inhibitory activity (TECA) have been discovered, obtained from enzymatic hydrolyzates of whey proteins (WO01 / 85984, Enzymatic treatment of whey proteins for the production of antihypertensive peptides, the resulting produets and treatment of hypertension in mammals), caseins (US6514941, Method of preparing a casein hydrolysate enriched in anti-hypertensive peptides), etc. Another group of bioactive peptides of great importance is that of peptides with anti-oxidant activity. Aging and various pathologies (neurological alterations, cancerous processes, cataracts, etc.) are related to the oxidation of cellular components, such as lipids, proteins or DNA, so the inclusion of antioxidants in the diet has a preventive character. In addition, their biological action, these compounds can prevent the oxidation of fats, avoiding the appearance of unpleasant flavors in food. Some research has revealed the ability of different proteins and hydrolysates to exert an antioxidant action, either acting as free radical scavengers (K. Suetsuna, H. Ukeda and H. Ochi, Isolation and characterization of free radical scavenging activities peptides derived from casein, Journal of Nutritional Bioche istry, 2000, 11: 128-131) or by inhibiting enzymes related to fat oxidation (SG Rival, S. Fornaroli, CG Boeriu and HJ Wichers, Caseins and casein hydrolysates. I Lipoxygenase inhibitory properties, Journal of Agricultural and Food Chemistry, 2001, 49: 287-294). It should be noted that studies of the structure-activity relationship of PECA peptides and antioxidants have revealed a characteristic common to both: the importance of certain amino acids hydrophobic in these two biological activities (HM Chen, K. Muramoto, F. Yamauchi, K. Fujimoto and K. Nokihara, Antioxidative properties of histidine-containing peptides designed from peptide fragments found in the digests of a soybean protein, Journal of Agricultural and Food Chemistry, 1998, 46: 49-53). Among the most used strategies for obtaining active peptides for food use, enzymatic hydrolysis and fermentation processes stand out. On the other hand, different technological treatments for protein functionalization have been proposed in recent years. The use of high hydrostatic pressures, as a physical method of denaturation, results in the modification of the non-covalent bonds responsible for the structure of the proteins, resulting in conformational changes that lead to an unfolded state. Several studies have shown that proteolysis is accelerated and different hydrolysis products appear under high pressure conditions, compared to what happens at atmospheric pressure F. Bonomi, A. Fiocchi, H. Fraki-er, A. Gaiaschi, S. Iametti, C. Poesi, P. Rasmussen, P. Restani and P. Rovere, Reduction of immunoreactivity of bovine β-lactoglobulin upon combined physical and proteolytic treatment, Journal of Dairy Research, 2003, 70: 51-59). In addition, it should be taken into account that most enzymes maintain their activity up to 400 MPa, inactivating at higher pressures. However, the production of bioactive peptides under high pressure conditions has not been described in the literature. Unlike other food proteins, there are very few studies related to bioactive peptides derived from egg proteins, although these are a very important source of nitrogen in the diet. H. Fujita, K Yokoyama, and M. Yoshikawa (Classification and antihypertensive activity of angiotensin I-converting enzyme inhibitory peptides derived from food proteins, Journal of Food Science, 2000, 65: 564-569) found TECA activities in ovalbumin hydrolysates with pepsin and thermolysin, with ϊCso values (concentration that inhibits 50% of the enzyme activity) of 45.0 and 83.0 μg / ml respectively. These authors isolated six peptides with TECA activity from hydrolyzed with pepsin, with IC50 values of 0.4 to 15 μM, but none of them, except the LW dipeptide, showed antihypertensive activity in spontaneously hypertensive rats (SHR). It was postulated that these peptides would be substrates of ECA, but not true inhibitors, so would show apparent ACEI activities in the in vitro assay (H. Fujita and M. Yoshikawa, LKPNM: a prodrug-type ACE-inhibitory peptide derived from fish protein, Immunopharmacology, 1999, 44: 123-127). These observations show that the determination of the TECA activity in vitro, although it is a good starting point for work, cannot become the only selection criterion, since it does not take into account the physiological transformations in the organism that determine the bioavailability of the peptides (digestion and passage through the gastrointestinal barrier to reach the blood in active form). Two peptides with vasodilator activity have been described, which come from the same region of ovalbumin hydrolyzed with different enzymes. H. Fujita, H. Usui, K. Kurahashi and M. Yoshikawa (Isolation and characterization of ovokmin, a bradykinin Bl agonist peptide derived from ovoalbumin, Peptides, 1995, 16: 785-790) found that ovokinin, an isolated octapeptide of an ovalbumin hydrolyzate with pepsin (FRADHPFL), showed vasorelaxive activity in canine mesenteric arteries, but did not have TECA activity. Ovokinin had antihypertensive activity when administered orally to spontaneously hypertensive rats (SHR) at doses of 100 mg kg (H. Fujita, R. Sasaki and M. Yoshikawa, Potentiation of the antihypertensive activity of orally administerrd ovokinin, a vasorelaxing peptide derived from ovalbumin, by emulsification in egg phosphatidyl-choline, Bioscience Biotechnology and Biochemistry, 1995, 59: 2344-2345). N. Matoba, H. Usui, H. Fujita and M. Yoshikawa (A novel anti-hypertensive peptide derived from ovalbumin induces nitric oxide-mediated vasorelaxation in an isolated SUR mesenteric artery, FEBS Letters, 1999, 452: 181-184), purified from a ovalbumin hydrolyzate with chymotrypsin, a hexapeptide corresponding to fragment 2-7 of ovokinin (RADHPF, ovokinin (2-7)) that exerted a potent vasodilator action in SHR at a dose of 10 mg / kg. Subsequently, ovokinin analogs (2-7) were synthesized in order to increase their antihypertensive activity. Among them, RPFHPF and RPLKPW, respectively, showed 10 and 100 times more activity than ovokinin (2-7) after oral administration to SHRs (minimum effective doses of 1 and 0J mg / kg), which was attributed to a higher Resistance to digestive tract proteases (N. Matoba, Y. Yamada, H. Usui, R. Nakagiri and M. Yoshikawa, Designing potent derivatives of ovokinin (2-7), an anti-hypertensive peptide derived from ovalbumin, Bioscience Biotechnology and Biochemistry, 2001, 65: 736-739 and Y. Yamada, N. Matoba, H. Usui and K. Onishi, Design of a highly potent anti-hypertensive peptide based on ovokinin (2-7), Bioscience Biotechnology and Biochemistry , 2002, 66: 1213-1217). It should be noted that, according to these authors and unlike most of the antihypertensive peptides of food origin, neither ovokinin (2-7), nor their RPFHPF or RPLKPW derivatives would possess TECA activity. It has been postulated that they would lower blood pressure through their interaction with gastrointestinal tract receptors or due to effects on the central nervous system. Given the high biological quality of egg proteins, it is of great interest to obtain, from them, bioactive peptides consumed as part of the diet, in addition to exercising their basic nutritional functions, are capable of producing metabolic or physiological effects, useful in health maintenance. The production of bioactive peptides from egg white proteins would allow new uses to be found in the chicken egg, beyond its classic nutritional value, including the production of medicinal and nutraceutical bioproducts. This would contribute to the development of healthy, safe and high quality foods, contributing to the use and revaluation of egg products.

DESCRIPTION OF THE INVENTION Brief description of the invention The present invention consists in the production of egg products containing bioactive peptides with JJECA activity in vitro and / or antihypertensive activity and / or antioxidant activity, by enzymatic hydrolysis of egg proteins. Bioactive peptides are produced by hydrolysis of one or more proteins, peptides or fragments thereof, which contain the amino acid sequence of said bioactive peptides (preferably containing ovalbumin), using enzymes (preferably pepsin) and hydrolysis conditions that allow the breakdown of the protein chain in the right places for its release. They can also be obtained by chemical or enzymatic synthesis or by recombinant methods etc. Such peptides can be consumed as such, or from crude hydrolysates, low molecular weight concentrates or other active subfractions obtained by size separation methods or chromatographic methods. In addition to being part of food products, such hydrolysates, their fractions or peptides could also be part of pharmaceutical products. Thus, they could help in the treatment and prevention of diseases, particularly in the control of blood pressure. The invention extends the applications of egg proteins, contributing to their utilization and revaluation.

Detailed Description of the Invention The invention provides a method for producing bioactive peptides from egg white proteins. Said bioactive peptides are those identified with the amino acid sequences shown in the SEQ. ID. No. 1, SEQ. ID. No. 2, SEQ. ID. No. 3, SEQ. ID. No. 4, SEQ. ID. No. 5, SEQ. JO. No. 6, SEQ. ID. No. 7 and SEQ. ID. No. 8 (table 1), some of which have LECA activity in vitro and / or antihypertensive activity in vivo and / or anti oxidant activity. The starting material of the present invention would be any suitable substrate comprising one or more proteins or peptides, of animal, vegetable origin or from micro-organisms, containing the amino acid sequence of the bioactive peptides of interest (SEQ. ID. No. .1, SEQ. ID No. 2, SEQ. D. No. 3, SEQ. ID. _.. 4, SEQ. ID. No. 5, SEQ. ID. No. 6, SEQ. ID No. 7 and SEQ ID No. 8, table 1), preferably ovalbumin or egg white. Since they all belong to the ovalbumin sequence, it is obvious that any preparation containing ovalbumin or ovalbumin peptides or fragments of any size, alone or mixed with other proteins, could be used. For example: pure ovalbumin, egg white and whole egg in its different forms of presentation, egg products intended for hospitality and catering, dietary supplements for athletes or egg products for animal feed etc. Said starting material is dissolved or dispersed, at an appropriate concentration, in water or in a buffer solution, at a pH suitable for the action of the proteolytic enzyme. Any proteolytic enzyme capable of breaking the protein present in the starting material and providing the peptides of interest may be employed, but preferably pepsin at pH 2-0-3.0. Proteolytic microorganisms that carry out fermentation of the substrate could also be used. The hydrolysis conditions: pH, temperature, pressure, enzyme-substrate ratio, reaction interruption etc., are optimized in order to select the most active hydrolysates. In a particular embodiment, the bioactive peptides are obtained using pepsin at pH 2.0, in a 1/100 enzyme / substrate ratio, w / w and performing hydrolysis at 37 ° C and atmospheric pressure (0J MPa), for a period of time between 10 min and 24 hours, but preferably for less than 3 hours. The use of high hydrostatic pressures, up to 400 MPa, accelerates the hydrolysis of the substrate without inhibiting the proteolytic enzyme and modifies the profile of the peptides obtained. Next, it is desired to concentrate the bioactive peptides and given that peptides with TECA activity contain approximately 3 to 6-7 amino acids (A. Pihlanto-Leppálá, T. Rokka and H. Korhonen, Angiotensin-I-converting enzyme inhibitory peptides derived from bovine milk proteins, International Dairy Journal, 1998, 8: 325-331), low molecular weight fractions can be obtained from hydrolysates by methods such as ultrafiltration, dialysis, electrodialysis with pore size membranes suitable, gel filtration chromatography, etc. In a particular embodiment, fractions of molecular weight less than 3000 Da of the hydrolysates are obtained by ultrafiltration through a hydrophilic membrane of 3000 Da. These fractions show greater ACEI and antihypertensive activity than the starting hydrolysates. From the low molecular weight fractions of the hydrolysates, active subfractions can be isolated by hydrophobic interaction chromatography, ion exchange chromatography or, preferably reverse phase high efficiency chromatography. In addition to the complete hydrolysates and their fractions, the peptides shown in Table 1 and indicated with the SEQ. ID. No. 1, SEQ. ID. No. 2, SEQ. ID. No. 3, SEQ. ID. No. 4, SEQ. ID. No. 5, SEQ. ID. No. 6, SEQ. ID. No. 7 and SEQ. ID. No. 8, possess bioactive properties, fundamentally TECA and / or antihypertensive and / or antioxidant activity and are also the subject of the present invention. Specifically, the peptides identified with the SEQ sequences. ID. No. 2, SEQ. ID. °. 3, SEQ. ID. No. 5 and SEQ. ID. J °. 6 show potent EECA activity in vitro and SEQ sequences. ID. No. 2, SEQ. ID. No. 3 and SEQ. ID. No. 6 possess antihypertensive activity in rats spontaneously hypertensive (SHR), but not in normotensive rats Wistar-Kyoto (WKY) when administered orally. On the other hand, at least the peptide identified as SEQ. ID. No. 6, has antioxidant activity against free radicals. It should be noted that these are natural peptides from which few side effects and good tolerance can be expected. Likewise, the bioactive peptides identified in the hydrolysates (SEQ. ID. No. 1, SEQ. ID. No. 2, SEQ. ID. No. 3, SEQ. ID. No. 4, SEQ. ID. No. 5, SEQ. ID. No. 6, SEQ. ID. No. 7 and SEQ. ID. No. 8) can be obtained by chemical and / or enzymatic synthesis of peptides or by recombinant methods.

Table 1. Sequences of the bioactive peptides identified YQIGL SEQ. ID. No. 1 IVF SEQ. ID. No. 2 RADHPFL SEQ. ID. No. 3 FSL SEQ. ID. No. 4 FRADHPFL * SEQ. ID. No. 5 YAEERYPIL SEQ. JX>. No. 6 RDTLNQ SEQ. ID. No. 7 SALAM SEQ. ID. No. 8

* Sequence previously identified as ovokinin (H. Fujita; H. Usui; K.

Kurahashi and M. Yoshikawa, Isolation and characterization of ovokinin, a bradykinin Bl agonist peptide derived from ovoalbumin, Peptides, 1995, 16: 785-790). It should be noted that these authors checked their vasodilator activity in canine mesenteric arteries, but affirm that they do not have TECA activity.

The obtaining of bioactive peptides from egg white hydrolysates with pepsin had not been previously described, although the JECA activity in vitro of ovalbumin hydrolysates (H. Fujita, K Yokoyama, and M. Yoshikawa, had already been demonstrated) Classification and antihypertensive activity of antiotensin I-converting enzyme inhibitory peptides derived from food proteins, Journal of Food Science, 2000, 65: 564-569). Egg white is a cheap and easily available protein substrate to produce bioactive peptides. On the other hand, there was no demonstrated the in vivo antihypertensive activity of ovalbumin hydrolysates. As already explained, it should be noted that frequently, many peptides that are shown as potent ACE inhibitors in vitro lose all or part of their activity when they are tested in vivo, or even, peptides that in vitro do not show great activity. as ACE inhibitors they acquire it in vivo due to the performance of digestive enzymes (M. Maeno, N. Yamamoto and T. Takano, Identification of an anti- hypertensive peptide from casein hydrolysate produced by a proteinase from Lactobacillus helvetics CP790, Journal of Dairy Science, 1996, 79: 1316-1321). These egg products: complete hydrolysates, their low molecular weight fractions, or one or more of their constituent bioactive peptides (including their derivatives, pharmaceutically acceptable salts and mixtures thereof), could be used as therapeutic substances with ACEI activity and / or with antihypertensive activity and / or antioxidant activity. Said egg products can be subjected to heat treatment, such as pasteurization, or undergo drying or lyophilization etc., to be used as functional food products, food additives or ingredients, or pharmaceutical products, for the treatment and / or prevention of hypertension. arterial in all its forms, mainly in humans, but also in animals. The amount of hydrolyzate, low molecular weight fraction, peptides, their derivatives or pharmaceutically acceptable salts and mixtures thereof, as well as their dosage for the treatment of any pathology will vary depending on numerous factors, such as age, severity of the pathology or dysfunction, route of administration and dose frequency. These compounds could be presented in any form of administration, solid or liquid, and administered by any appropriate oral, respiratory, rectal or topical route, although they are particularly designed for solid or liquid administration by oral route. In general, the process of obtaining these egg products: the complete hydrolysates, the low molecular weight fractions thereof and their constituent peptides, can be optimized, directing it to the production of the greatest possible amount of bioactive peptides or to control possible the appearance of bitterness, normally caused by a high concentration of hydrophobic peptides of intermediate or low molecular weight. Analytical procedures Measurement of angiotensin converting enzyme (ACEI) inhibitory activity Angiotensin converting enzyme (ACE) inhibitory activity is measured in vitro according to the DW Cushman and HS Cheung (Spectrophotometric assay and properties of the method) angiotensin-converting enzyme of rabbit lung, Biochemical Pharmacology, 1971, 20: 1637-1648), subsequently modified by YK Kim, S. Yoon, DY Yu., B. Lonnerdal and BH Chung (Novel angiotensm-I-converting enzyme inhibitory peptides derived from recombinant human O _s i-casein expressed in Escherichia coli. Journal of Dairy Research 1 99, 66, 431-439). The histidyl leucine hippyl substrate (BHL, Sigma, Chemicals Co, St. Louis, MO, USA) is dissolved in 0.1 M borate buffer with 0.3 M NaCl, pH 8.3, to obtain a final concentration of 5 mM. To 100 μl of substrate are added 40 μl of each of the samples whose LECA activity is to be determined. ECA enzyme (EC 3.4.15.1, Sigma), dissolved in 50% glycerol, and diluted at the time of the 1/10 test in double-distilled water is added. The reaction is carried out at 37 ° C, for 30 minutes in a water bath. The enzyme is inactivated by lowering the pH with 150 μl of 1N HC1. The formed hippuric acid is extracted with 1000 μl of ethyl acetate. After vortexing for 20 seconds, it is centrifuged at 4000 rpm for 10 min and at room temperature. 750 µl of the organic phase is evaporated by heating at 95 ° C for 10 minutes. The hippuric acid residue is redissolved in 800 μl of double distilled water and, after stirring for 20 seconds, the absorbance at 228 nm is measured on a Dur-70 spectrophotometer from Beckman Instruments, Inc., Fullerton, USA. The following formula is used to calculate the percentage of ACEI:

Acontrol-Amuestra% IECA = * 100 Acontrol-Ablanco

White is used to correct background absorbance. This contains substrate, enzyme and 20 μl of double distilled water instead of sample, and the reaction is stopped at zero time. The control assumes one hundred percent of the enzymatic action on the substrate in absence of inhibitors, and contains 20 μl of water instead of sample and incubates the same time as the sample. The results are presented as IC ₅₀ (μM or μg / ml) or concentration at which 50% of enzyme activity is inhibited. The protein concentration is determined by the bicinconinic acid (BCA) assay (Pierce, Rockford, JL, USA) using bovine serum albumin as the standard.

Measurement of antioxidant activity To measure antioxidant activity, the method developed by R Re, N. Pellegrini, A. Proteggente, A. Pannala, M. Yang and C. Rice-Evans (Antioxidant activity applying an improved ABTS) is used radical cation decolorization assay, Free Radical Biological Medicine, 1999, 26: 1231-1237). The method is based on the disappearance of the ABTS ^{* +} radical (2,2-azinobis (3-ethylbenzothiazoline-6-sulfonic acid) due to the reducing action of different samples with antioxidant activity. The ABTS ^{* 4 "} radical has a maximum of absorbance at 734 nm. The antioxidant activity is detected by the decrease in absorbance time of 734 nm. To generate the ABTS radical " ⁺ , the compound ABTS (Sigma), dissolved in water at a concentration of 7 mM, is put into 2.45 mM potassium persulfate contact in a 1: 2 ratio for 24 hours avoiding light After 24 hours, the absorbance at 734 nm is adjusted to a value of 0.70 ± 0.02 with 5 mM saline phosphate buffer (PBS), 138 mM NaCl, pH = 7.4 In a typical experiment, 1 ml of this solution is added to 10 μl of the sample (1.5 mM), dissolved in 0.02 M sodium phosphate buffer, pH = 6.5. in triplicate, incubating each sample with the ABTS ^{* +} radical for 10 minutes at 37 ° C, making readings of absorbance at 734 nm in minute 1, 6 and 10. As a blank, 0.02 M sodium phosphate buffer, pH = 6.5 is used and at the same time that the samples are incubated, the ABTS ^{* +} radical is incubated without agent antioxidant (control). The antioxidant activity of each sample is calculated with respect to the absorbance at 734 nm presented by the control. The antioxidant activity of the sample is expressed in relation to the antioxidant activity of Trolox (6- hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid) (Sigma), a vitamin E analogue, using a TEAC value (antioxidant capacity equivalent to Trolox) at a certain time. Different ways of calculating the TEAC value have been developed. We use the method described by FWPC van Overlveld, GRMM Haenen, J. Rhemrev, JPW Vermeiden and A. Bast (Tyrosine as important contributor to the antioxidant capacity of seminal plasma, Chemical and Biological Interactions, 2000, 127: 151-161), which It is valid to calculate the TEAC value of a pure compound, which causes a decrease in absorbance that increases linearly with increasing concentration. In this case, it would only be necessary to calculate a certain concentration of a compound in order to calculate its TEAC value. Variation of Abs ₇₃ nm l-CΑ -'- 'Compound 0.28045 x [Compound concentration]

The value 0.2845 represents the decrease in absorbance caused by 1 mM of Trolox.

Isolation of peptide fractions by high-performance liquid chromatography in reverse phase (RP-HPLC) on a semi-preparatory scale A device consisting of two programmable pumps model Waters Delta 600, an aligned diode detector model 966, an automatic injector model is used

717 plus, and an automatic fraction collector (Waters Corp., Mildford, MA, USA)

A C ₁₈ Prep NovaPack® HR column, 7.8 x 300 mm and 6 μm pore size is used

(Waters), with a Cι ₈ cartridge (Waters) as a column guard. The analyzes are performed at

30 ° C and detection at 214 and 280 nm. Data acquisition is carried out with the Millennium Software version 3.2 (Waters). For the elution of the samples a binary gradient of water (phase A) and acetonitrile (phase B) with 0J% TFA in each of them and a flow of 4 ml / min is used. The phase B gradient is 2% to 10% in 15 min, from

10% to 20% for 35 min, from 20% to 30% for 20 min, the column is washed with

70% B and finally it is conditioned in the initial conditions for 15 min. The volume of injected sample is 200 μl. Before injection, the samples are passed through a 0.45 μm Millipore (Waters) filter. Analysis using tandem mass spectrometry (off-line) An Esquire 3000 ion trap unit is used (Bruker Daltonik GmbH, Bremen, Germany). The sample is injected into the electrospray nebulizer at a flow of 4 μl / min, using a syringe pump type 22 (Harvard Apparatus, South Natick, MA, USA). The equipment uses nitrogen as a nebulizer and drying gas, and operates with a helium pressure of 5 x 10 ^{~ 3} bar. Mass spectra are acquired in a range of 50-1500 m / z and at a speed of 13000 Da / second. The interpretation of the MS spectra in tandem for the identification of the peptide sequences is carried out with the Biotools 2.1 program (Bruker Daltonik GmbH, Bremen, Germany).

Analysis by RP-HPLC coupled to online mass spectrometry in modem (RP-HPLC-MS / MS) An Esquire-LC device (Bruker Daltonik GMBH, Bremen, Germany) is used. The HPLC equipment (1100 series) consists of a quaternary pump, an automatic injector, an eluent degassing system and a variable wavelength ultraviolet detector (Agilent Technologies, Waldbronn, Germany) coupled in line to a mass spectrometer of ionic trap Esquire 3000 (Bruker Daltonik). The column is a Hi-Pore C18 column (250 x 4.6 mm di., 5 μm particle size) (Bio-Rad Laboratories, Richmond, CA, USA). Solvent A is a mixture of water and trifluoroacetic acid (1000: 0.37) and solvent B a mixture of acetonitrile and trifluoroacetic acid (1000: 0.27). 50 μl of sample are injected at a concentration of 1-2 mg / ml. A flow of 0.8 ml / minute is used, with a linear gradient from 0% to 30% of solvent B in A in 25 minutes. The eluent is monitored at 214 nm, by mass spectrophotometry, under the same conditions as indicated in the previous section, except that the injection flow of the sample through the nebulizer is 60 μl / min.

Study of antihypertensive activity in spontaneously hypertensive rats The effect of several of the identified peptides and some products containing them (for example, egg white hydrolyzate with pepsin for 3 hours and the fraction thereof less than 3000 Da) is studied. about blood pressure of spontaneously hypertensive (SHR) and Wistar-Kyoto (WKY) rats, which are the normotensive control of SHR. This study is carried out with SHR (10) and WKY, aged 17-24 weeks and weighing between 300 and 350 g, from Charles River Laboratories Spain SA Rats are stored in cages five by five and kept at a temperature stable at 25 ° C with 12-hour light-dark cycles, ingesting water and food freely available. To perform systolic blood pressure (SBP) measurements, a tail cuff device (Le5001, Letica) is used that provides a digital value of the SBP automatically and records and facilitates the heart rate of the Animals Several measurements are made, and the average of all of them is obtained to obtain a reliable value of the PAS Before placing the sleeve and the transducer in the tail of the rats, these are exposed to a temperature close to 30 ° C to facilitate dilation of the caudal artery In addition, to ensure the reliability of the measurement, the animals get used to the procedure 2 weeks before carrying out the test in question.The administration of the products to be tested is carried out by intragastric tube in a period of time between 9 and 10 am The SHR used for the study had PAS values between 230 and 280 mm Hg. The WKY used for the study at that time they had PAS values between 160 and 200 mm Hg. Measures of the PAS of the animals are taken periodically, every 2 hours, until 8 hours post-administration of the products to be tested; additionally, a measure of the PAS is taken 24 hours after the administration of said products. As a negative control (to establish the circadian variation of the SBP in probed rats) the SBP measurements obtained in rats that are administered by intragastric tube 1 ml of water are used. As a positive control, the SBP measures obtained in rats that are administered by intragastric tube 50 mg / kg of Captopril (prototype drug TECA) are used. This dose of Captopril is administered to each rat in a volume of 1 ml. To establish the effect of non-hydrolyzed egg white on the blood pressure of the animals, tests similar to those described above are carried out, in which the animals are treated by the same procedure with 200 mg / kg of egg white previously lyophilized (control). The results obtained are grouped and the mean ± the standard error of the mean (ESM) is obtained for a minimum of 9 homogeneous tests. The data of the treated animals are always compared with the data considered negative control. To compare them and obtain statistical significance, the "t" test of "Student" is used for unpaired data and the difference for values of p <0.05 is considered significant.

Brief description of the content of the figures Figure 1 represents the decrease in systolic blood pressure (SBP), obtained in spontaneously hypertensive rats, after administration by intragastric tube of 1 ml of water (o), 50 mg / kg of Captopril ( π), 200 mg / kg of egg white (CH) (X), and different doses of egg white hydrolyzate (HCH): 100 mg / kg

(), 150 mg / kg (A), 200 mg / kg (m), and 400 mg kg (Δ). Data represent the mean ± ESM for a minimum of 9 animals. The abscissa axis represents the time, in hours, elapsed since the administration. Figure 2 represents the decrease in systolic blood pressure (SBP), obtained in spontaneously hypertensive rats, after administration by intragastric tube of 1 ml of water (o), 50 mg / kg of Captopril (D) and different doses of fraction less than 3000 Da of egg white hydrolyzate (F <3000 Da): 25 mg / kg (4), 50 mg / kg (A) and 100 mg / kg ("). Data represent the mean ± ESM for a minimum of 9 animals The time in elapsed time since administration is represented on the abscissa axis Figure 3 A is a chromatogram obtained using reverse phase high efficiency liquid chromatography (RP-HPLC) on a preparative scale of the fraction less than 3000 Da produced by hydrolysis of egg white with pepsin for 3 h, in which 9 fractions (F1-F9) are selected, which were automatically collected.The time in minutes is represented on the abscissa axis. Figure 3 B represents the TECA capacity, expressed as the con Protein centering necessary to inhibit 50% of the enzyme (IC50), corresponding to each of the 9 fractions collected by RP-HPLC on a preparative scale. Figure 4 represents the decrease in systolic blood pressure (SBP), obtained in spontaneously hypertensive rats, after administration by tube. intragastric of 1 ml of water (o), 50 mg / kg of Captopril (D) and different doses of the YAEERYPΓL peptide: 0.5 mg / kg (i), 1 mg / kg (A) and 2 mg / kg (u). Data represent the mean ± ESM for a minimum of 9 animals. On the abscissa axis, the time, in hours, transcribed from the administration is represented. Figure 5 represents the decrease in systolic blood pressure (SBP), obtained in spontaneously hypertensive rats, after administration by intragastric tube of 1 ml of water (o), 50 mg / kg of Captopril (π) and different doses of the peptide RADHPFL: 0.5 mg / kg (4), 1 mg / kg (A) and 2 mg / kg (m). Data represent the mean ± ESM for a minimum of 9 animals. The abscissa axis represents the time, in hours, elapsed since the administration. Figure 6 represents the decrease in systolic blood pressure (SBP), obtained in spontaneously hypertensive rats, after administration by intragastric tube of 1 ml of water (X), 50 mg / kg of Captopril (π) and different doses of the peptide INF: 1 mg / kg (Φ), 2 mg / kg (A) and 4 mg / kg (u). Data represent the mean ± ESM for a minimum of 9 animals. The abscissa axis represents the time, in hours, elapsed since the administration. Figure 7 represents the variation of systolic blood pressure (SBP), obtained in normotensive Wistar-Kyoto rats, after administration by intragastric tube of 1 ml of water (o), 50 mg / kg of Captopril (D), 200 mg / kg of CH (X), 400 mg / kg of HCH (•), 100 mg kg of the fraction less than 3000 Da of HCH (F <3000 Da) (Δ), 2 mg / kg of the YAEERYPEJ peptide », 2 mg / kg of the RADHPFL peptide (A) and 4 mg / kg ΓVF peptide (B). Data represent the mean ± ESM for a minimum of 9 animals.

The abscissa axis represents the time, in hours, elapsed since the administration.

EXAMPLES OF EMBODIMENT OF THE INVENTION The following examples illustrate the invention, although they should not be considered as limiting the scope thereof,

Example 1. Obtaining bioactive peptides, with ACEI and antihypertensive activity, from egg white hydrolyzed with pepsin at atmospheric pressure. The hydrolyzate was obtained using as a clear substrate of chicken egg, from fresh eggs, separated from the yolk and lyophilized. . As an enzyme, pepsin (EC 3.4.23.1. Type A, 10000 U / mg protein), from pork stomach (Sigma) was used. The substrate was dissolved in water at a concentration of 100 mg / ml and the pH was adjusted to 2.0 by adding 1N HC1. Pepsin (substrate enzyme ratio 1/100, w / w) was added. Hydrolysis was performed at a temperature of 37 ° C for 24 hours, at atmospheric pressure (0.1 MPa). Pepsin inactivation was achieved by raising the pH to 7.0 with 1N NaOH. JECA activity was measured in aliquots collected after different hydrolysis times, 0, 30 minutes, 3, 5, 8 and 24 hours. The results showed that the egg white without hydrolyzing does not have ACEI activity (IC50> 750 μg / ml), but actively inhibits ACE after different hydrolysis times with pepsin, reaching an important inhibition after 3 hours of hydrolysis (ICso = 200.9 ± 1.5 μg / ml; 55.3 ± 2.1 μg / ml; 72.2 ± 2.3 μg / ml; 43.07 ± 1.4 μg / ml; 40.2 ± 0.9 μg / ml; at 30 min, 3, 5, 8 and 24 hours, respectively) . The fraction less than 3000 Da of the egg white hydrolyzate with pepsin for 3 hours was obtained by ultrafiltration through a hydrophilic membrane of 3000 Da (Centriprep, Amicon, Inc., Beverly, MA, USA), centrifuging at 1900 g for 40 minutes. ACEI activity was measured in the retained (size fraction> 3000 Da) and in the permeate (size fraction <3000 Da). The IC ₅₀ values were, respectively, 298.4 and 34.5 μg / ml. This shows that the permeate has approximately 10 times more JECA activity and that, therefore, the activity is mainly due to small peptides. The antihypertensive activity of the egg white hydrolyzate with pepsin was tested for 3 hours and its fraction less than 3000 Da in spontaneously hypertensive rats (SHR) and in Wistar-Kyoto (WKY) normotensive rats. Different concentrations of both lyophilized egg products were administered to the animals that, in the in the case of the hydrolyzate, they were between 100 and 400 mg / kg, and in the case of the fraction less than 3000 Da, between 25 and 100 mg / kg. Figures 1 and 2 show, respectively, the decrease in SBP obtained in SHR at different times after administration of different doses of egg white hydrolyzate with pepsin for 3 hours, and after administration of different doses of the smaller fraction of 3000 Da of said hydrolyzate. The results of these tests with non-hydrolyzed egg white (control) (200 mg / kg) are shown in Figure 1. It can be seen that the PAS values corresponding to the control are similar to the PAS values of animals a Those who administer water. These figures also include the decrease in SBP observed after the administration of Captopril. Captopril produces a pronounced decrease in SBP in SHR. The decrease in SBP is maximum 6 hours after drug administration. The egg white hydrolyzate and the fraction less than 3000 Da cause significant dose-dependent decreases of SBP in animals. The lowest PAS values after administering the egg white hydrolyzate and the fraction less than 3000 Da of this hydrolyzate, are also observed 6 hours after administration. The SBP values observed 24 hours after the different administrations are similar to those that the animals had before them. In order to identify the peptides responsible for the PECA and / or antihypertensive activity, the fraction less than 3000 Da obtained after hydrolysis of egg white with pepsin for 3 hours was lyophilized and redissolved in water at a concentration of 50 mg / ml and It was fractionated by RP-HPLC on a semi-preparative scale. As shown in Figure 3A, 9 fractions were collected (after approximately 10-12 analyzes), which were frozen, lyophilized and kept at -20 ° C until use. Each fraction was redissolved in milli-Q water and IECA activity was measured. As shown in Figure 3 B, the most active subfractions were 6, 7 and 8, which were analyzed by tandem mass spectrometry to determine their constituent peptides. To that end, the peptide subfractions 6, 7 and 8, collected by preparative RP-HPLC, were lyophilized and dissolved at a concentration of 5-10 μg / ml in a 50% acetonitrile mixture in water containing 0.3% acid formic. The identified peptides are shown in Table 2. It should be noted that all peptides came from ovalbumin. Table 2. Peptides identified in subfractions 6, 7 and 8 of the fraction. less than

3000 Da of the egg white hydrolyzate with pepsin for three hours Mass Dough

Fraction. 11th. Protein Position Amino acids Sequence theoretical experimental number 6 592.3 592.32 üvoalbúrπina 212-210 YQIGL SEQ. ΪD. ¥ . i 6 377.2 377.23 Ovσalbúrnina 178-180 l ^' V ^' F SEQ. ΪD. No. 2 6 854.4 854.44 Ovoalbú-nina 359-365 RADHPFL SEQ. ΪD. No. 3 6 365.2 365.20 Gvoa ina 99-101 FSL SEQ. ΪD. No. 4 7 721.3 721.37 Ovalbumin 256-26 and ESÜNF 7 1001.4 1001.51 Ovalbumin 358-365 FRADHPFL SEQ. ID. No. 5 7 1152.3 1152.58 Ovalbumin 106-1 14 YAEERYPΠ. I KNOW THAT. ID. No. 6 8 757.2 757.41 Ovalbumin 84-89 RDILNQ SEQ. ID. No. 7 8 1040.2 1040.58 Ovalbumin 243-252 V LPDEVSGL 8 491.1 491.24 Ovalbumin 36-40 SALAM SEQ. ΪD. W. 8 8 487.1 487.26 Ovoalbú ina 144-147 EI.IN 8 1164.2 i] 04.59 Ovoalbúniina 125-134 Y ^' RGGLEPÍNF

EjeoipI © 2. Peptides obtained by chemical synthesis with TECA and antifaipertensive activity All identified peptides, mentioned in Table 2 of Example 3, were chemically synthesized using the solid phase Fmoc method with a 43 JA model synthesizer from Applied Biosystems Inc. ( Überlingen. Germany). The purity of the synthetic peptides was verified by RP-H.PI.C ~ MS MS. The IECA activity of synthetic peptides was measured. The activity found is shown in Table 3. 8 peptides with ICJO lower than 450 μM stand out for their activity and, above all, the sequences: SEQ. THE. No. 2, SEQ. ID. No. 3, SEQ. ID. No. 5 and SEQ. ID. No. 6, with IC ₅₀ less than 34 μM.

Table 3. BECA activity of the peptides identified in subsections 6, 7 and 8 of the fraction less than 3000 Da hydrolyzed egg white with pepsin for three hours Sequence No. Amino Acids IC ₅₀ (μM) SEQ ID No. 1 VQIGL ¡ 73 8 SLQ K ° 2 JVF 33 9 SEQ ID ° 3 RΛDHPFJ 6 2 SEQ ID No. 4 FSL 172 9 fcSUNJ "> 1000 SEQ ID No. 5 RADJIPFL 3 2 SEQ ID No. 6 YAEF.RYPIL 4 7 SEQ ID. No. 7 RDILNQ 435 7 VLLPDEVSGL> 1000 SLQ m. S ° 8 SAIAM 229 1 F.LIN> 1000 YRGGLEPTNF> 1000 The antihypeitensive activity of peptides SEQ lü 3NP 2, SLQ ID was tested

N ° 3 v SEQ. ID N ° 6, for which different doses of the same were administered to SHR and WKY, the maximum dose used being always equivalent in units of SECA activity at the dose 50 mg / kg of the less than 3000 Da of the hydrogenated egg claia The popados were dissolved in distilled water and the corresponding dose was administered to each rat in a volume of J mi Las. Figiuas 4, 5 and 6 show the decreases in SBP obtained in SHR at different times, after administration of different doses of the peptides SEQ ID No. 6, SBQ JD No. 3 and SEQ ID No. 2 It can be seen that the administration of these peptides causes a significant dose-dependent decrease in SBP in these animals. The decrease in Ja PΛS is maximum 6 hours after the administration of these peps. and the maximum decrease obtained is also similar with the different peptides. Figure 7 shows the changes of the PAS obtained in WKY ralas at different times, after the administration of the following compounds 400 mg / kg of the egg white hydrolyzate, 100 mg / kg of the Menoi fraction of 3000 Da of hydrolyzed, 2 mg / kg of the SEQ.LD.N ⁰ peptide. 6.2 mg / kg peptide SEQ.ID.N ⁰ . 3, and 4 mg / kg peptide SEQ.JJD.N ⁰ . 2. The results obtained after administration of 50 mg / kg of Captopril are also included. It can be seen that none of these compounds modifies the PAS of WKY rats when the highest dose used is administered. These results allow us to rule out possible undesirable effects of the products tested on the blood pressure of normotensive subjects. The results presented demonstrate that the peptides identified by the SEQ sequences. ID. No. 2, SEQ. ID. No. 3 and SEQ. ID. No. 6, have a clear and pronounced antihypertensive effect, which after its acute administration follows a temporary course similar to the antihypertensive effect that is seen when the egg white hydrolyzate or the fraction less than 3000 Da of said hydrolyzate is administered.

Example 3. Peptides obtained by chemical synthesis with antioxidant activity The anti-oxidant activity of one of the identified peptides, specifically the SEQ sequence, was measured. ID. No. 6, mentioned in example 1. The activity found is shown below: TEACγAEERYPIL (l minute) = 0.8 TEACYAEERYPI (6 minutes) = 1.2 TEACYAEERYPIL (l O minutes) ⁼⁼ 1.3

The results show, therefore, that 1 mg of YAEERYPJL (SEQ. ID. No. 6) has 1.3 times more antioxidant activity than 1 mg of Trolox.

Example 4. Obtaining bioactive peptides from ovalbumin hydrolyzed with pepsin at atmospheric pressure The hydrolyzate was obtained using ovoalbumin substrate grade VI (99% purity) (Sigma). The substrate was dissolved in water at a concentration of 100 mg / ml and the pH was adjusted to 2.0 by adding 1N HC1. In this particular embodiment of the invention, pepsin (EC 3.4.23.1. Type A, 10000 U / mg protein), from pork stomach (Sigma) was added at an enzyme / substrate ratio 1/100, w / w. Hydrolysis was carried out at a temperature of 37 ° C for 3 hours, at atmospheric pressure (0.1 MPa). Pepsin inactivation was achieved by raising the pH to 7.0 with 1N NaOH. The measurement of JECA activity showed that unhydrolyzed ovalbumin does not have JECA activity (IC ₅₀ > 750 μg / ml), but inhibits the enzyme after 3 hours of hydrolysis with pepsin (IC50 = 129.0 ± 0.6 μg / ml). The hydrolyzate thus obtained was analyzed by RP-HPLC-MS / MS. At least the sequences were found: SEQ. JO. No. 1, SEQ. ID. No. 2, SEQ. ID. No. 3, SEQ. ID. No. 4, SEQ. ID. No. 5, SEQ. ID. No. 6, SEQ.

ID. No. 7 and SEQ. ID. No. 8. Among them, the SEQ sequences. ID. No. 2, SEQ. ID. No. 3,

I KNOW THAT. ID. No. 5 and SEQ. JD No. 6 have IC50 lower than 34 μM (example 2). SEQ.

ID. No. 2, SEQ. ID. No. 3 and SEQ. THE. No. 6 also show antihypertensive activity in rats (example 2), and SEQ. ID. No. 6 has antioxidant activity against free radicals (example 3).

Example 5. Obtaining bioactive peptides from ovalbumin hydrolyzed with pepsin under conditions of high hydrostatic pressure. The hydrolyzate was obtained using ovoalbumin substrate grade VI (99% purity) (Sigma). As an enzyme, pepsin (EC 3.4.23.1. Type A, 10000 U / mg protein), from pork stomach (Sigma) was used. The substrate was dissolved in water at a concentration of 2 mg / ml and the pH was adjusted to 2.0 by adding 1N HC1. Pepsin (enzyme / substrate ratio 1/20, w / w) was added. The hydrolysis was carried out at a temperature of 37 ° C for 30 min, at different hydrostatic pressures (100, 200, 300 and 400 MPa). Pepsin inactivation was achieved by raising the pH to 7.0 with 1N NaOH. The treatments with high pressure were carried out in a discontinuous hydrostatic pressure equipment (900 HP Eurotherm Automation) with a capacity of 2350 ml, which reaches a pressure of 500 MPa. The high-pressure chamber consists of a stainless steel cylinder, filled with the pressure-transmitting medium (water), into which the substrate and enzyme mixture is introduced, packed in an Eppendorf plastic tube without leaving an air chamber. The equipment reaches the desired pressure at a speed of 2.5 MPa / second and, after treatment, drops to zero at the same speed. The equipment is accompanied by an auxiliary bath that, through the circulation of water through an outer jacket that surrounds the cylinder, allows treatments at temperatures from -20 ° C to 95 ° C. The process temperature is controlled by a thermocouple submerged in the pressure transmitting medium. The hydrolysates thus obtained were analyzed by RP-HPLC-MS / MS. At least the sequences were found: SEQ. ID. No. 3, SEQ. ID. No. 5 and SEQ. ID. No. 6 which, as indicated in example 2, have IC50 of less than 7 μM. SEQ. ID. No. 3 and SEQ. ID. No. 6 also show antihypertensive activity in rats (example 2), and SEQ. ID. No. 6 has antioxidant activity against free radicals (example 3). This example shows how the use of high hydrostatic pressures allows hydrolysates containing active peptides to be obtained more quickly than by hydrolysis at atmospheric pressure. It should be noted that the SEQ was not obtained under these conditions. ID. No. 2, which could have an impact on the industrial applicability of hydrolysates.

Claims

1. Bioactive product identified from enzymatic hydrolysis of egg white proteins characterized by a. have ACEI activity in vitro and / or antihypertensive activity in vivo and / or antioxidant activity. b. have a molecular weight between 365.2 and 1152.58 c. be peptide identified with the amino acid sequences of the following group: SEQ. ID. No. 1, SEQ. ID. No.

2, SEQ. ID. No. 3, SEQ. ID. No. 4, SEQ. ID. No. 5, SEQ. ID. No. 6, SEQ. DD. No. 7 and SEQ. ID. No. 8 2. Bioactive product identified from enzymatic hydrolysis of egg white proteins according to claim 1, characterized in that it is obtained by a chemical or enzymatic synthesis process or by recombinant methods.

3. Method for producing bioactive products according to claim 1 and 2 characterized by a, obtained by hydrolysis of the starting material that would be any suitable substrate containing one or more proteins or peptides, of animal, vegetable origin or from rhino-organisms, preferably ovalbumin or egg white whose amino acid sequence comprised the amino acid sequence of any of the bioactive peptides of interest indicated in claim 1, b. dissolving or dispersing said starting material, at an appropriate concentration, in water or in a buffer solution, at a pH suitable for the action of the proteolytic enzyme, c. any proteolytic enzyme capable of breaking the protein present in the starting material and providing the peptides of interest, but preferably pepsin at pH 2-0-3.0; or proteolytic microorganisms that will carry out a fermentation of the substrate, and d. the reaction time would be between 10 min and 24 hours, but preferably for less than 3 hours.

4. Process for producing bioactive products according to claim 3 characterized by using high hydrostatic pressures between 100 and 1000 MPa, preferably 400 MPa, to accelerate the hydrolysis of the substrate without inhibiting the proteolytic enzyme and / or modifying the profile of the peptides obtained

5. Bioactive product identified from erizimatic hydrolysis of egg white proteins characterized in that the procedures indicated in claims 3 and 4 are used to obtain them,

6. Bioactive product identified from enzymatic hydrolysis of egg white proteins according to claim 5, characterized in that the starting material is, among others, pure ovalbumin, egg white and whole egg in its different forms of presentation, or intended products to hospitality and catering, dietary supplements for athletes or egg products for animal feed.

7. Bioactive product identified from enzymatic hydrolysis of egg white proteins according to claim 6 characterized in that it is the enzymatic hydrolyzate, any of its actions, or a purification thereof containing at least some of the indicated peptides in claim 1

8. Bioactive product identified from enzymatic hydrolysis of egg white proteins characterized by being pharmaceutically acceptable derivatives or salts or mixtures thereof of any of the bioactive products indicated in claims 1,2,6,6 and 7.

9. Pharmaceutical composition characterized by comprising at least some of the bioactive products with TECA activity in vitro and / or antihypertensive activity in vivo and / or antioxidant activity according to claims 1,2,6,6 and 7.

10. Additive, ingredient or functional food supplement characterized by comprising at least some of the bioactive products with ACEI activity in vitro and / or antihypertensive activity in vivo and / or antioxidant activity according to claims 1,2,6,6 and 7.

11. Functional food product characterized by comprising at least some of the bioactive products with ACEI activity in vitro and / or antihypertensive activity in vivo and / or antioxidant activity according to claims 1,2,6,6 and 7.

12. Use of the pharmaceutical composition according to claim 9 in the preparation of a medicament for the treatment of hypertension.

13. Use of additive, ingredient, functional food according to claim 9 in the preparation of a favorable functional food product to reduce hypertension.